CALRETICULIN BIOSYNTHESIS AND PROCESSING IN HUMAN MYELOID CELLS - DEMONSTRATION OF SIGNAL PEPTIDE CLEAVAGE AND N-GLYCOSYLATION

Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium . Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to it s biosynthesis, particularly in human tissues. We report analyses of s ynthesis, proteolytic processing and glycosylation of human calreticul in, In both HL-60 and PLB-985 myeloid cell lines calreticulin was immu noprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, w as processed by cotranslational signal peptide cleavage to a 60-kD spe cies that comigrated with mature calreticulin produced in myeloid cell s, Neither tunicamycin treatment of the cells nor endoglycosidase dige stion of calreticulin resulted in any forms other than the 60-kD prote in on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analys is, suggesting that the potential site for N-glycosylation at asparagi ne-327 was unmodified, However, oxidative derivatization of carbohydra te components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indisti nguishable electrophoretic mobilities, Direct measurement by phenol-H2 SO4 confirmed the presence of carbohydrate on recombinant human calret iculin, These data show that human myeloid calreticulin undergoes cotr anslational signal peptide cleavage and posttranslational N-linked gly cosylation, Although glycosylation of calreticulin has been shown in r at liver and bovine liver and brain, it has been reported to be lackin g in other tissues including human lymphocytes. (C) 1997 by The Americ an Society of Hematology.